Cyclic replication of the of the Escherichia coli chromosome from oriC requires a biochemical oscillator that generates a periodic signal for the initiation of replication. Current models of initiation control assume that DnaA protein, product of the dnaA gene, gradually accumulates at multiple binding sites, either at oriC or elsewhere on the chromosome, and triggers replication initiation when it has reached a certain threshold concentration. This concentration is always reached at a certain cell mass per oriC, called "initiation mass". Although DnaA overproduction may, under certain conditions, trigger an extra round of replication, we observed that continued overproduction under normal growth conditions has no effect on the initiation mass. From these and other observations, we have argued that DnaA is probably not directly involved in the oscillator mechanism generating the cell cycle (Bremer and Churchward, 1991; and unpublished work from this laboratory). It is therefore proposed to begin a systematic search for other genes and sites that might be involved in the control of replication initiation. For this purpose, methods have been developed to select bacterial mutants with smaller or larger cell size. Since the initiation mass is one of the factors determining the cell mass and size, it is expected that mutants with altered initiation mass will be found among such cell size variants. Preliminary experiments showed this expectation to be justified. The genetic map location of the mutations in a collection of such mutants with altered initiation mass will be determined to find the genes involved. New genes will be cloned and the effect of their over- or underexpression studied, to identify potential candidates for factors that drive the cell cycle "clock", as a first step to understand its underlying mechanism.